A simple and effective method for identifying inhibitors of protein-protein interactions(PPIs)was developed by using capillary electrophoresis frontal analysis(CE-FA).Anti-apoptotic B-cell-2(Bcl-2)family member Bcl-XL protein,a 5-carboxyfluorescein labelled peptide truncated from the BH3 domain of Bid(F-Bid)as the ligand,and a known Bcl-XL-Bid interaction inhibitor ABT-263 were employed as experimental model for the proof of concept.In CE-FA,the free ligand is separated from the protein and protein-ligand complex to permit the measurement of the equilibrium concentration of the ligand,hence the dissociation constant of the protein-ligand complex.In the presence of inhibitors,formation of the protein-ligand complex is hindered,thereby the inhibition can be easily identified by the raised plateau height of the ligand and the decayed plateau of the complex.Further,we proposed an equation used to convert the IC50 value into the inhibition constant Ki value,which is more useful than the former for comparison.In addition,the sample pooling strategy was employed to improve the screening throughput more than ten times.A small chemical library composing of synthetic compounds and natural extracts were screened with the method,two natural products,namely demethylzeylasteral and celastrol were identified as new inhibitors to block the Bcl-XL-Bid interaction.Cell-based assay was performed to validate the activity of the identified compounds.The result demonstrated that CE-FA represents a straightforward and robust technique for screening of PPI inhibitors.